The betulinic acid derivative IC9564 inhibits human immunodeficiency virus (HIV)-1 entry. Among a series of IC9564 derivatives, 5 and 20 were the most promising compounds against HIV infection with EC(50) values of 0.33 and 0.46 microM, respectively. Both compounds inhibited syncytium formation with EC(50) values of 0.40 and 0.33 microM, respectively. The comparable EC(50) values in the two assays suggested that these compounds are fusion inhibitors. The structure-activity relationship data also indicated that a double bond in IC9564 can be eliminated and the statine moiety can be replaced with L-leucine while retaining anti-HIV activity.